Project Summary This proposal aims at the development of a platform for the comprehensive classification and characterization of post-transcriptional modifications (PTMs) in non-protein coding RNAs (ncRNAs). The availability of a convenient approach for the detection of N6-methyladenosine (m6A), which relies on specific antibodies and RNA-seq, has enabled groundbreaking studies that revealed the significance of this PTM in essential regulatory processes. In particular, seminal reports on viral replication and cocaine addiction have clearly shown that m6A pathways are very promising targets for the development of new therapeutic strategies. The implementation of a more versatile approach based on mass spectrometry allowed us to show that the genome of many RNA viruses is decorated by different types of RNA modifications in addition to m6A. We also found that long non-coding RNAs involved in stress response and cancer contain constellations of ribonucleotide variants. Based on the m6A precedents, we anticipate that elucidating their roles in the activities of the respective parent RNAs will open countless new avenues for therapeutic intervention. This project will develop tools for determining the incidence and distribution of PTMs, which is essential for their functional elucidation. Libraries of antisense DNA-probes will be employed to capture desired classes of RNAs identified by genetic screens, which will be immediately analyzed for PTM content. Following a divide-and-conquer strategy, the libraries will target progressively narrower pools to enable the classification of PTM-bearing RNAs. The multi-fold sample enrichment afforded by the capture process will allow the isolation and concentration of individual RNAs to be submitted to mass mapping and sequencing. The sample preparation steps will be carried out on microtiter well plates to multiplex the entire process, reduce sample losses, support unattended operations, minimize the duration of each analysis, and eliminate any delay between consecutive analyses. The platform development will initially employ standards consisting of synthetic and recombinant RNAs, which will be promptly replaced with actual biological samples from different cellular systems. The latter will primarily consist of long non-coding RNAs obtained from human monocytes and yeast cultures under different stress conditions. The results will provide new precious insights into the role of PTMs in the stress response mediated in humans and yeast by the homologous p38-MAPK and HOG pathways, respectively. The enabling technologies developed here will immediately benefit several ongoing collaborations in the fields of genetics, epigenetics, human virology, and cancer biology. The ability to take these projects in new unimaginable directions substantiates the excellent innovative impact of these technologies. These projects are representative of much broader communities with an enormous stake in understanding the effects of PTMs on the structure and function of their parent RNAs. For this reason, these technologies will be poised to become an essential research and diagnostic tool for any health condition involving RNA regulation, including different types of cancers, neurological deficits, developmental malformations, growth and mental retardation, mitochondrial disorders, and susceptibility to viral infection and stress.